DE69830135T2 - Vector containing Mycobacterium tuberculosis 38 kDa antigen for the treatment of neoplasms - Google Patents

Abstract

The Mycobacterium tuberculosis Ag38 gene, which encodes a highly immunogenic protein, was cloned into a retroviral vector in-frame with the leader and the transmembrane portion of the nerve growth factor receptor, and transduced into murine melanoma cell line B16-B78. Significant protection was observed in mice immunized with the transduced melanoma cells and subcutaneously challenged with parental melanoma cells since only 20% of mice developed tumors. Necroscopy of mice immunized with the transduced melanoma cells revealed dramatic inhibition of experimental metastases induced by intravenous (i.v.) inoculation of parental melanoma cells. Moreover vaccination with transduced cells significantly prolonged survival of mice challenged i.v. with parental melanoma cells. These data indicate that the presence of the mycobacterial 38-kDa protein greatly enhances immunological recognition of structures expressed by the parental melanoma cells. Comparison of Th1 and Th2 responses in mice immunized with parental melanoma cells versus mice receiving the transduced cells revealed a clear predominance of Th1 responses when the Ag38 protein was endogenously expressed. This novel transduction approach based on the 38kDa antigen may represent a promising immunotherapeutic strategy for the treatment of cancer patients. In addition, as vaccine the transduced cells may be used for inducing immunity to cancer. <IMAGE>

Description

Recent studies have identified cell surface antigens on mice and human tumors of various histotypes that are potential targets for an immune response. These antigens, which are recognized by host T cells, are derived from endogenously synthesized proteins that arise either through mutations in malignant transformation or through abnormal expression of genes that are normally silent in adult tissue. However, these antigens are usually very small immunogens in spontaneously developing tumors. In efforts to elicit an immune response against these tumor antigens, various microorganisms, in particular Mycobacterium bovis bacillus Calmette-Guérin (BCG), have been used as immunoadjuvant in patients with various cancers, but the results have been varied. ² Currently, patients with superficial bladder cancer are treated with an immunotherapy consisting of intravesical instillation of BCG. There is some evidence that a similar mechanism is underlying the efficacy of BCG in tumor prevention and therapy ³ and in the prevention of tuberculosis. This efficacy appears to depend on whether a predominant Th1 or Th2-type cytokine secretion pattern is induced. ²

In another attempt to stimulate an immune response against tumors, various cytokine genes were introduced into tumor cells. The secreted cytokines were found to induce a protective immune response against the tumor. ⁴ The administration of manipulated tumor cells, interleukin-2 (IL-2), IL-4, interferon-gamma (IFN-γ) or express granulocyte-macrophage colony stimulating factor (GM-CSF), shows that they locally and often systemically induce immunity. However, elimination of existing tumors has been less frequently achieved. ⁵ In some cases, however manipulated cytokine-expressing tumor cells show an increased metastatic potential. ⁶

Further, prior art ^{18 describes} the transformation of the murine macrophage tumor cell line J774 with the highly immunogenic hsp65 of Mycobacterium leprae and concludes that the loss of carcinogenicity is achieved by this method by enhancing recognition of the tumor-associated antigens. Further reference is made to the transformation of murine tumor cells ²⁷ , namely the thymoma cell line EL4 and the lymphoma line RMA with a plasmid encoding Ag38, pXJ38. The transformation of these cells with Ag38, however, is only for the purpose of forming cells useful for inducing a lymphocytotoxic response to tubercle bacilli following mouse inoculation, and there is no suggestion of any kind for the use of Ag38 vectors for the treatment of neoplasms. Finally, reference is made to a review article on therapeutic vaccines for melanomas, which review also concerns BCG vaccines.

Since the combination of poorly immunogenic and highly immunogenic determinants on a common carrier can increase the immune response against the low antigenic determinants by stimulating immune recognition ⁷ , the invention addresses the issue of whether immune recognition of malignant cells can be increased by transduction of tumor cells a gene encoding a Mycobacterium protein is thought to represent a danger signal because it elicits a strong T cell response. ⁸ when the expression of this gene in an increased maturation of Th1 cells can also be induced against tumor cells, antigens, a T-cell response and this may finally, in the selective destruction of tumor cells by a specific anti-tumor immune response result. According to the invention, the murine melanoma cell line B16-B78 was transduced with a Mycobacterium tuberculosis (strain H37Rv) gene encoding a 38 kDa protein (Ag38), ⁹ this is one of the strongest immunogens of the Mycobacterium tuberculosis antigens. ^Ten transduced cells were injected into mice and the immune response against parental B16-B78 melanoma cells was examined.

In Referring to the known 38 kDa antigen of Mycobacterium tuberculosis and homologs thereof may be referred to e.g. EP-A 92 108,254.1 and the references cited therein.

Of the inventive expression vector may be a vector in which the polypeptide is wild-type or recombinant 33 or 38 kDa antigen of Mycobacterium tuberculosis is.

Of the inventive expression vector may be a virus, especially an inactivated retrovirus, a retroviral shuttle vector, such as pLTMSN, a vaccinia virus Adeno and / or a plasmid, and / or the nucleic acid can Be RNA.

Farther can the expression vector of the invention to be an episomal vector.

Farther can the expression vector of the invention include a sequence containing an antigen associated with neoplasms encodes and / or a sequence encoding an interleukin.

According to one another embodiment The invention relates to the use of a cell, in particular a neoplasmic cell line, in particular a human cell or Cell line containing an expression vector of the invention.

Farther For example, the invention relates to a cell or virus that is included in their Genomes a heterologous nucleic acid, which encodes a polypeptide as defined above, in expressible form contains.

A cell according to the invention or cell line may further comprise an expression vector having a Sequence encoding a neoplasm associated antigen included.

A cell line according to the invention or cell may further comprise an expression vector having a sequence, which encodes a neoplasm-associated antigen or an interleukin, contain or include.

A another embodiment The invention relates to the use for the production of a pharmaceutical Composition comprising the expression vector, the cell, the Cell line or virus according to the present invention Invention and a pharmaceutically acceptable carrier, such as a liposome.

The Pharmaceutical composition may further include an expression vector with a sequence containing a neoplasm associated antigen or an interleukin encoded.

The Pharmaceutical composition can be an effective, non-toxic Amount of expression vector, cell, cell line or virus, as described above.

According to the invention Expression vector, the cell line, the cell, the virus or the Nucleic acid for Producing a drug used to treat neoplasms, being treatment for existing neoplasms or prophylactically can be done.

A Discussion of the embodiment the invention follows.

Carcinogenicity of transduced Melanoma cells

A modified retroviral vector (pLTMSN) containing the human nerve growth factor receptor (NGFR) leader and transmembrane portion and the human IL-3 stop codon region was constructed. The DNA encoding residues 24-372 of the Mycobacterium tuberculosis Ag38 gene excluding the leader peptide and the stop codon were cloned in-frame to obtain the pLAg38TMSN vector ( 1 ). This modified vector expresses the 38 kDa antigen on the cell surface, allows the detection and tagging of transduced cells by an anti-Ag38 monoclonal antibody (MAb), HBT12. ⁹ The supernatant from the producer cell lines was tested for viral titer and antigen expression by FACScan analysis with MAb HBT12 and used to infect murine B17-B78 melanoma cells. ¹¹ transduced cells expressed the Mycobacterium tuberculosis Ag38 transcript and produced the bacterial protein as determined by RT-PCR ( 2a ) or Western Blot ( 2 B ) was detected. Reactivity with MAb HBT12 revealed two bands in whole cell membrane extract at 47 kDa and 42 kDa, respectively, corresponding to the unprocessed form containing both the leader and transmembrane sequences and the mature protein after removal of the leader sequence. The majority of the transduced B16-B78 cells were positive for cell surface expression of the mycobacterial protein as determined by FACS analysis ( 2c ). Both wild-type and melanoma cells transduced with the vector that does not contain the Ag38 gene did not show any detectable mycobacterial product by RT-PCR, Western blot or FACS analysis.

The carcinogenicity of the parenteral melanoma cell line and the cells transduced with the 38 kDa gene was tested in C57BL / 6 mice. All five mice were injected sc with 2.5 x 10 ⁵ B16-B78 cells, developed tumors (average 13.8) within 9 to 21 days, and had an average survival time of 36.6 days. Nekroskopie showed lung metastases in all of these mice. In contrast, three out of five mice injected with 2.5 x 10 ⁵ transduced melanoma cells developed tumors, and these mice showed a delay in tumor incidence (averaging 30.6 days), resulting in an increase in survival time (mean 52.3 days); no metastases were found in the lungs, liver, kidneys or abdomen. Two other 38 kDa transduced clones, which are available in unab also showed no spontaneous metastases (not shown), this makes it unlikely that the Ag38 gene was inserted into a gene involved in the metastasis.

Ag38-transduced melanoma cells confer systemic immunity against parenteral melanoma cells

In the experiments to determine whether immunization with transduced melanoma cells protects mice against subsequent provocation with parenteral cells ( 3 ), all 9 naive mice and 8 out of 10 mice immunized with wild-type melanoma cells developed tumors, while significant protection (p <0.01) was observed in mice immunized with transduced melanoma cells because only 2 out of 10 mice developed tumors. Furthermore, tumor progression and mortality were delayed in these mice compared to unimmunized and wild-type melanoma immunized animals. Because the wild-type melanoma cells are highly carcinogenic and poorly immunogenic, these data indicate that expression of the 38 kDa antigen in transduced cells produces a strong immune response to the tumor. Endogenous expression of Ag38 appears to be essential for protection because C57BL / 6 mice immunized with purified 38 kDa protein developed tumors at the same rate as unimmunized controls when challenged with wild-type melanoma cells were provoked.

Experimental metastases induced by iv inoculation with 5 x 10 ⁵ wild-type melanoma cells were inhibited in mice immunized with irradiated Ag38-transduced cells, demonstrated by the absence of a small number of lung metastases 4 weeks after challenge compared to control mice ( 4a ). Two experiments were performed using the same provocation protocol to examine the effect of immunization on survival time. In the first experiment ( 4b ), the 6 mice immunized with Ag38 transiently survived significantly longer than the 6 unimmunized control mice (p = 0.0005), all developed lung metastases and died within 31 days after tumor inoculation. Two out of 6 immunized mice were still alive at the end of the observation period (90 days). In the second experiment, a significant survival increase (p = 0.0001) in the 8 immunized mice was observed over the 8 control mice, 2 animals were still alive after 150 days. These data demonstrate that vaccination with murine melanoma cells transduced with the 38 kDa Mycobacterium gene induces significant inhibition of lung metastases from intravenously injected parenteral tumors, resulting in prolonged survival.

Pattern of cytokine secretion of Th1 and Th2 type

Due to the increasing evidence suggesting the importance of the Th1-like response in controlling tumor growth, the Th1 and Th2 type response was examined in mice, which was injected into the footpad with i) irradiated wild-type melanoma cells ii) irradiated transduced cells and iii) irradiated wild type melanoma cells plus 38 kDa purified protein. As shown in Table 1, analysis of cytokine levels in the culture supernatants of lymphocytes obtained from ipsilateral popliteal lymph nodes shows an increase in IFN-γ levels in the mice injected with transduced cells compared to the mice irradiated with wild-type melanoma Cells were injected and to mice injected with irradiated wild type melanoma cells plus 38 kDa purified protein. In contrast, the secreted IL-4 levels were higher in the mice injected with wild-type B16 plus purified 38 kDa protein than in the supernatant of lymphocytes from animals injected with transduced cells (data not shown). Induction of the preferred Th1 cytokine secretion pattern by endogenously expressed mycobacterial antigen is consistent with the study of Huygen et al. ¹³ and Zhu et al. Figure ¹⁴ , which shows that the vaccinations with plasmid DNA constructs encoding the 85T or 38 kDa Mycobacterium tuberculosis antigen are effective approaches to generating a specific helper response with a Th1-like phenotype.

As previously described ¹⁵ , the B78 variant of melanoma B16 expresses undetectable levels of major histocompatibility complex (MHC) class I antigens in vitro, even after exposure to IFN-γ (not shown). Low expression of class I antigens often characterizes advanced tumors and metastases in humans. ¹⁶ Indeed, the down-regulation of MHC class is an important mechanism 1 expression by the tumors T-cell mediated immune responses avoid. The expression of the 38 kDa protein on the surface of the B16-B78 melanoma cell can not stimulate MHC-restricted cells, such as γ / δT cells, which then influence the initial activation of the subset of Th-1 cells.

Regardless of the MHC context, a recent report suggested that the immunological Intervention with molecules that selectively activate Th-1 responses may be effective in activating NK cells, cytotoxic T lymphocytes, and tumoricidal macrophages, thus enhancing killing of the tumor cells. ¹⁷

The enhancement of the immune response to tumor antigens was achieved by transducing tumor cells with two different genes encoding the heat shock protein (HSP) 65 from Mycobacterium leprae ¹⁸ and Mycobacterium bovis. ¹⁹ HSPs are molecular chaperones that aid in the assembly and folding of other proteins. ²⁰ HSPs are associated in vivo with the entire repertoire of cell-derived peptides, and these non-covalent HSP-peptide complexes are particularly immunogenic. ²¹ In the present study we have shown that the expression of a Mycobacterium tuberculosis protein, which no chaperone shows ^22, the immunogenicity of poorly antigenic tumor cells lacking co-stimulatory molecules and no detectable levels of MHC antigens express reinforce.

This Transduction approach can be a promising immunotherapeutic Strategy to treat cancer.

A Detailed description of the invention with reference to the figures and the experimental data follows.

description the figures

1

schematic Representation of the pLAg38TMSN retroviral vector. LTR = Moloney murine leukemia virus long terminal repeat; ψ = packaging signal; Ag38 = Mycobacterium tuberculosis Ag38 gene; SV = SV40 previous promoter; NEO = neomycin phosphotransferase gene. The arrows show the start of transcription at. Not to scale drawn.

2

expression of Ag38 and transduced cells. A) Detection of Ag38 mRNA by RT-PCR of total RNA from: B16-B78 Wild-type melanoma cells (lane 2); B16-B78 cells transduced with pLTMSN vector (lane 3); B16-B78 cells transduced with pLAg38TMSN vector (lane 4); and pLAg38TMSN vector (Lane 5). Lane 1 molecular marker (1 kb ladder). B) Evidence of Ag38 protein with Western blot analysis of the membrane proteins extracted from: B16-B78 wild-type melanoma cells (Lane 2); B16-B78 cells transduced with pLTMSN vector (lane 3); and B16-B78 cells transduced with pLAg38TMSN vector (Lane 4). Lane 1 bacterial recombinant Ag38 protein. C) proof of Ag38 protein by FACScan analysis on the surface of B16-B78 Zllen transduced with pLTMSN vector (top) and B16-B78 cells transduced with pLAg38TMSN vector (below). Open spaces identify cells labeled with the secondary antibody alone.

3

Percent tumor incidence (A) and overall survival (B) of mice either unimmunized (dotted line) or immunized with 10 ⁶ irradiated wild-type B16-B78 (dashed line) or irradiated transduced B16-B78 melanoma cells (solid line). The mice were challenged with 2.5 x 10 ⁵ wild-type B16-B78 melanoma cells.

4

Lung colonization and overall survival of mice provoked iv with 5x10 ⁵ wild-type B16-B78 melanoma cells. A) Lung metastases of mice immunized with irradiated transduced B16-B78 melanoma cells (top) and unimmunized mice (bottom). B) Survival of immunized mice (dashed line) and unimmunized mice (solid line).

Examples

retroviral construct

One SacII-PvuII fragment containing the first 843 base pairs of the receptor Nerve Growth Factor (NGFR), which is the intracellular domain was absent, was cloned in a pGEM 5Zf + (Promega); the extracellular area of the NGFR's became cut out with stuI and avaI and in frame with a synthetic Polynucleotide sequence containing three unique cloning sites (NotI, ClaI and EcoRV) (referred to as pLTM). At the 3'-end the last 28 bases of the human IL-3 sequence containing the stop codon in frame by replacing the NcoI-NheI IL-3 fragment with the SacII-SpeI fragment of pLTM in the pBBG14 vector (British Biotechnology; Oxon, UK). The whole construct was cut from the plasmid (HindI-BamHI) and into the HpaI-BamHI sites of the retroviral vector pLXSN alloyed (kindly by Dr. D. Miller; Fred Hutchinson Center, Seattle, Washington, U.S.A.). The whole Vector, referred to as pLTMSN, was constructed using the Sanger method (Sequenase Version 2, USB Amersham Life Science, Cleveland, Ohio, U.S.A.).

The DNA encoding residues 24-372 of Mycobacterium tuberculosis Ag38 was obtained by PCR using plasmid pMS9-2 ⁹ as a template and two primers with modified NotI and ClaI sites at the 5 'and 3' ends, respectively. The sense primer was 5'-GCGGCCGCTGGCTCGAAACCACCGAGCGGTTCGCCT GAA-3 'corresponding to nucleotides 215-246 of the Ag38 gene and the antisense primer was 5'-GCGGTGGTGAAGTTGTCTGACGCGTTGATCGCATC-GATT-3' corresponding to nucleotides 1235-1267. The PCR was carried out with 30 cycles at one minute and 94 ° C, one minute and 55 ° C and two minutes at 72 ° C. The PCR product was digested with NotI and ClaI as restriction enzymes and ligated in frame to the corresponding sites of the pLTMSN retroviral vector to obtain the pLAg38TMSN vector ( 1 ).

Retroviral particles were treated with infection-described techniques obtain gp ²³ by using the ampholytic ecotropic packaging cell lines + AM12 and GP + E86. ^24.25 G418-resistant isolated colonies of producer cells were screened for surface expression of Ag38 expression by FACScan analysis using anti-Ag38 MAb HBT12. ⁹ The viruses prepared from the best packaging cell line, expressed as viral titer and antigen expression, were used to infect murine melanoma cells.

transduction and expression of the Mycobacterium gene in cells

B16-B78 murine melanoma cells ¹¹ (6 x 10 ⁵ ) were cultured for 2 hours with 5 ml undiluted viral supernatant of the packaging cell line and 8 μg / ml polybrene; fresh medium was then added for the next 24 hours to dilute the supernatant (1: 2). In order to improve the infection efficiency, the same infection cycle was repeated 5 times. Isolated colonies as well as bulk colonies of G418-selected (1 mg / ml) melanoma cells were tested for 38 kDa antigen gene expression by RT-PCR, FACScan analysis and Western blot.

G418-resistant colonies derived from B16-B78 melanoma cells transfected with the empty vector were used as controls. For the RT-PCR (QIAGEN GmbH, Hilden, Germany Rneasy ^® Handbook,) were obtained from 1 x 10 ⁷ cells total RNA; 1 μg of the RNA was transcribed (GeneAmp Rna PCR Kit, Perkin Elmer Cetus, Norwalk, Connecticut, USA) and the cDNA was amplified by PCR using the same 5 'primer as used for cloning and a 3' primer ( 5'-TGATCGCCCAGTGCAGAAAT-3 ') corresponding to nucleotides 1265-1284. Thirty cycles of amplification (one minute at 94 ° C, one minute at 55 ° C and two minutes at 72 ° C) were performed and the reaction product (950 base pairs) was electrophoresed and stained with ethidium bromide.

The Flow cytometry has been used to detect the cell surface antigen used. Briefly, the cells were purified for 30 minutes at 4 ° C with 10 μg / ml incubated anti-Ag38 MAb HBT12, washed and incubated for a further 30 minutes at 4 ° C Goat anti-mouse IgG + IgM (H + L) FITC-conjugated secondary antibody (Kirkegaard & Perry Laboratories Inc .; Gaithersburg, MD, USA). After washing, the Cells with a FACScan (Becton Dickinson, Mountain View, CA, USA) analyzed.

For Western blot analysis, a membrane protein extract according to Landowsky et al. ^Obtained with minor modifications: 1.5 x 10 ⁸ cells were in ice-cold 25 mM Tris / 0.3 M sucrose (pH 7.4) buffer including protease inhibitor by ultrasonication with 5 x 5 s bursts on ice, followed by centrifugation at 500 g for 10 minutes at 4 ° C. This step was repeated three times and the supernatant was collected. The membrane fraction was recovered by centrifugation of the supernatant at 143,000 g for 90 minutes at 4 ° C. The pellet was resuspended and rotated end over end for 12-16 hours at 4 ° C in 50 mM Tris / 150 mM NaCl, pH 7.4 buffer containing 1% NP40 detergent and protease inhibitor. Insoluble material was pelleted by centrifugation at 200,000g for 60 minutes, and the supernatant was electrophoresed on a 15% reducing SDS-PAGE gel. The proteins were detected with MAb HBT12 as the primary antibody and peroxidase-coupled secondary antibody visualized using the ECL detection method (Amersham).

In vivo experiments

Female C57BL / 6 mice, 6 to 8 weeks old, were obtained from Charles River (Calco, Italy). All mice were according to the guidelines treated and held.

The carcinogenicity of the cells was tested in mice injected subcutaneously (sc) with 2.5 x 10 ⁵ transduced or parenteral tumor cells into the right side. For immunization, 10 ⁶ transduced or parenteral irradiated (20,000 rad) melanoma cells sc were injected into the left side of 20 mice (10 animals per group, twice at a 4-week interval). Four weeks after the second immunization, mice were challenged sc in the right flank with 2.5 x 10 ⁵ wild-type melanoma cells. Nine control mice received only the challenge with living cells. The mice were examined twice weekly for tumors and two rectangular diameters of tumor mass were recorded.

In experiments to test a vaccination protocol against experimental lung metastases, the mice were immunized with irradiated cells, followed by an iv inoculation of 5 x 10 ⁵ live parenteral melanoma cells. The control mice received only the iv inoculation. The mice were sacrificed four weeks thereafter to determine the number of lung metastases. The same experimental protocol was used in two experiments to study survival with 6 and 8 animals per group, respectively. The differences between the groups were analyzed with the log-rank test.

• *Daten beziehen sich auf einen representatives Experiment, das im dreifachen Ansatz durchgeführt wurde und ausgedrückt als Durchschnitt ± SD erhalten nach Abzug des Cytokin-Niveaus im Überstand von Lymphozyten, die in Abwesenheit eines anti-CD3-Antikörpers kultiviert wurden.
• IL-4/IFN-γ-Verhältnis zwischen B16 transduziert zu B16 p = 0,0013; B16 transduziert zu B16 + Ag38 p = 0,0109 (unpaariger T-Test).

In experiments to study the pattern of cytokines released by T cells, mice (6 per group) sc in the right hind footpad were irradiated with 5 x 10 ⁶ transduced or non-transduced irradiated cells or 3 μg Ag38 purified protein mixed with the non-transduced irradiated Immunize cells in 50 μl of saline. Five days later, the mice were sacrificed and the popliteal lymph nodes were removed aseptically. Pools were made for each group, each containing the nodes of two or three animals. The lymphocytes were mechanically loosened and cultured (2x10 ⁵ cells per well) in 96-well flat bottom plates either precoated with 1 μg per well of anti-CD3 monoclonal antibody or also not precoated at 37 ° C for 18 hrs. The cytokine-containing supernatant was collected and assayed for IFN-γ and IL-4 production by commercial ELISA kits (Genzyme, Cambridge, MA, USA). The unpaired T-test was used to determine the significance of differences between the IL-4 / IFN-γ ratio between the groups. Table 1

• * Data refer to a representative experiment performed in triplicate and expressed as mean ± SD obtained after subtraction of cytokine levels in the supernatant of lymphocytes cultured in the absence of anti-CD3 antibody.
• IL-4 / IFN-γ ratio between B16 transduced to B16 p = 0.0013; B16 transduced to B16 + Ag38 p = 0.0109 (unpaired T-test).

Another example

Around to determine if Mycobacterium tuberculosis protein also has immune recognition of other antigens that are not restricted to melanoma cells, To investigate, we examined another antigenic model.

This Model consists of a mouse transgenic for the rats newly proto-oncogene, added into the germ line under the MMTV promoter. These transgenic mice develop spontaneously mammary carcinomas after a latency of 6 months, this over-express the transgene encoded oncoprotein.

The c-erbB-2 human oncogene homologue of rat neu gene was found to be Immunogen in patients; therefore, this module was selected to to investigate whether the transduction of Mycobacterium tuberculosis protein which can enhance anti-neu immunogenicity.

Around the endogenous expression of the Mycobacterium tuberculosis protein To obtain, we have the N202-1A tumor cell line in vitro with the retroviral vector pLAg38TMSN transduced this tumor cell line is a cell line derived from a transgenic mouse mammary carcinoma , characterized by overexpression of the neu oncoprotein.

We have chosen a clone, the adequate one Levels of Mycobacterium antigen expressed on the cell surface and used this clone to perform some in vivo experiments.

Around the effect of in vivo vaccination with transduced cells To investigate induction of protection against spontaneous tumor development we have female mice twice with irradiated transduced or not transduced Cells immunized 2 to 4 months before the spontaneous tumor develops, and we have between the groups the growth of spontaneous focal Examined mammary tumors and compared.

In a preliminary one Have experiment with a small number of animals (5 per group) we observe that all were immunized with parenteral cells Mice spontaneously Tumors developed while partial protection in 2 out of 5 mice, which were immunized with transduced cells could (a delay in tumor development in an animal and the absence of the tumor in another animal). This result was due to the low Number of group not significant.

The same protocol was used to in a subsequent experiment a larger number of To immunize animals (11 per group). We observed tumor growth in all animals in both groups with a delay in 7 out of 11 mice, the were immunized with the transduced cells. Furthermore showed the mice immunized with transduced cells a smaller and smaller Number of tumors.

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Claims (22)

Use of an expression vector for the production of a pharmaceutical for the treatment of neoplasms, wherein the vector nucleic acid includes, including at least one sequence expressing a polypeptide coded, this is i) as a 38 kDa antigen of Mycobacterium tuberculosis known antigen or ii) a 33 kDa protein, which is a derivative of i) with N-terminal deletion, the Protein does not contain the signal sequence, or only 17 to 24 codons contains the signal sequence, or not the signal sequence and another 24 amino acids at Contains N-terminus. Use according to claim 1, wherein the expression vector is a virus. Use according to claim 2, wherein the nucleic acid RNA is. Use according to claim 3, wherein the expression vector is an inactivated retrovirus. Use according to claim 3, wherein the expression vector is a retroviral shuttle vector. Use according to claim 5, wherein the expression vector pLTMSN, as in 1 shown is. Use according to claim 2, wherein the expression vector is a vaccinia virus. Use according to claim 2, wherein the expression vector is an adenovirus. Use according to claim 1, wherein the expression vector is a plasmid. Use according to claim 1, wherein the expression vector is an episomal vector. Use according to one the previous claims, further containing a sequence associated with neoplasms Encoded antigen. Use according to one the previous claims, wherein the expression vector further comprises a sequence whose interleukin encoded, contains. Use of a cell containing an expression vector according to one the previous claims contains, to Preparation of a pharmaceutical for the treatment of neoplasms. Use of a neoplastic cell line, wherein their cells an expression vector according to one of claims 1 to 12 contains for the preparation of a pharmaceutical for the treatment of neoplasms. Use according to claim 13 or 14, wherein the cell is a human cell or cell line. Use according to one the claims 13-15, wherein the cell or cell line further contains an expression vector with a sequence for encodes a neoplasm-associated antigen. Use according to one the claims 13-15, wherein the cell or cell line further contains an expression vector with a sequence for an interleukin encoded contains. Use, which claimed according to one of the preceding claims Pharmaceutical agent further contains a pharmaceutically acceptable carrier. Use according to claim 18, the carrier is a liposome. Use according to claim 18 or 19, wherein the pharmaceutical agent further comprises an expression vector with a sequence for an antigen associated with neoplasms or an interleukin encoded, contains. Use of a nucleic acid according to claim 1 for the preparation a drug for the treatment of neoplasms. Use according to one the previous claims, the treatment being directed to existing neoplasms.